Abstract 16884: CircNPHP1 Regulates Endothelial Function in Ischemic Heart Disease

Abstract

Background: Ischemic heart disease (IHD), which is characterized by reduced myocardial blood flow, is aggravated by diabetes mellitus (DM). Non-coding RNAs, including microRNAs (miRs) and circular RNAs (circRNAs), regulate gene expression and impact angiogenesis. Canonically, miRs bind to the 3’UTR region of their target messenger RNAs (mRNA) to mediate post-transcriptional gene inhibition. CircRNAs can “sponge” and sequestrate miRs, thus preventing the repression of their mRNA targets. Hypothesis: The circRNAs/miR/mRNA interactions are deregulated in the diabetic heart, contributing to IHD. Aims: To identify and mechanistically investigate the circRNA-miRNA-mRNA networks deregulated in IHD and diabetes. Methods: We generated bulk RNA-seq data on whole transcriptome (for mRNAs and circRNAs) and small RNA (for miRs) using heart left ventricle biopsies of 3 groups of cardiac surgery patients (n=12/group) from the ARCADIA cohort (13/LO/ 1687): 1) and 2) suffering from IHD with/out type-2 DM and 3) non-ischemic non-diabetic controls. Customized bioinformatics approaches identified differentially expressed (DE) circRNAs, mRNAs and miR in IHD and IHD+T2DM patients (vs. Controls). Prediction tools and databases (CircInteractome, miRWalk) identified “sponging associations” between circRNAs and miRs and the targets of sponged miRs, producing the circRNA-miR-mRNA networks. The network’s individual components were filtered for endothelial cell (EC) expression using GEO datasets and for involvement in angiogenesis using KEGG, Wiki and Panther pathways and Gene Ontology. The endogenous role of the circRNAs in the top emerging network was tested in cultured ECs using siRNA-mediated silencing, while the circRNA-miRNA sponging interaction were confirmed determined after using circRNA pulldown with divergent primers. Results: A putative proangiogenic subnetwork commanded by circNPHP1 is DE in IHD+DM. circNPHP1 silencing in the ECs reduced angiogenesis and downregulated 2 validated target genes of miR-221-3p, namely VEGF and BCL2. The pull-down assay confirmed circNPHP1 binding to miR-221-3p. Conclusions: circNPHP1 supports vascular repair and holds potential as novel therapeutic target in IHD and DM.